The overall objective of this grant proposal is to investigate the role of individual adhesion molecules, as well as cytokines which modulate adhesion molecule expression, in the allergen mediated adhesion of eosinophils to endothelium in vitro and in vivo. In vivo studies of eosinophil adhesion to endothelium will utilize adhesion molecule and cytokine knockout mice to assess the relative importance of an individual adhesion molecule (P- selectin, E-selectin and ICAM) or cytokines which modulates adhesion molecule expression (TNF, IL-1) to the allergen mediated adhesion of eosinophils to endothelium. Fluorescently labelled mouse eosinophils will be injected into the tail vein of allergen challenged adhesion molecule/cytokine knockout or control mice and the rolling, adhesion and transmigration of the fluorescently labelled eosinophils compared in vivo in the two groups of mice using intravital videomicroscopy. The in vitro videomicroscopy studies of eosinophil adhesion to endothelium will use a novel single cell adhesion assay able to measure strength of adhesion in (mdynes) to determine whether juxtacrine cytokine signalling from endothelium to eosinophil modulates the function state of VLA-4 (as opposed to the number of VLA-4 receptors) to affect eosinophil binding to its counterreceptors (VCAM or CS-1) under static and flow conditions. FACS analysis and confocal laser scanning microscopy will determine whether eosinophil active cytokines alter VLA-4 receptor number of receptor distribution in association with changes in VLA-4 adhesive function. To determine whether endothelial lumenal expressed cytokines such as GM-CSF can function in juxtacrine signalling to eosinophils under conditions of flow we will determine whether endothelial cells express cell surface GM- CSF (as opposed to secreted GM-CSF) using immunohistochemistry, confocal microscopy and radiolabelled GM-CSF. The functional ability of endothelial expressed GM-CSF to signal to eosinophils will be assessed under conditions of flow using a parallel plate flow chamber. Overall, these studies should improve our understanding of the molecular mechanisms of eosinophil adhesion to endothelium in vitro and in vivo.